The overall objective of this research proposal is to delineate at the molecular level the different steps in poliovirus encapsidation. To guide our studies, we propose a model for encapsidation in which 3CD (a fusion between 3C and 3D) bound to the 5' nontranslated region of new plus strand RNAs chaperones the genome to the capsid precursor (P1); the RNA genome then compacts, facilitated by the helicase activity of 2C. The VPg protein interacts specifically with the poliovirus capsid proteins, most probably the amino terminus of VP1. Specific capsid amino acids interact with the RNA to facilitate encapsidation. To accomplish these studies, during the last funding period we established a system which disengages the replication process from the encapsidation process by constructing recombinant vaccinia viruses which express P1 and 3CD. We have also developed a system to encapsidate poliovirus genomes encoding foreign proteins (e.g. luciferase). The following Specific Aims are proposed: Specific Aim 1: To define the roles of 3CD and 2C in encapsidation. We will analyze mutations in 3C and 3D for the capacity to affect encapsidation. The role of 2C in encapsidation will be delineated by the development of a complementation system to provide 2C from a recombinant vaccinia virus. Specific Aim 2: To define amino acids of VPg required for replication and encapsidation. Using mutagenesis, we will delineate which amino acids of VPg are essential for encapsidation. Experiments will determine whether VPg specifically interacts with the amino terminus of VP1. Specific Aim 3: To delineate the critical amino acids of P1 and genomic RNA sequences required for interaction between the capsid and RNA genome. We will further define the role of specific capsid amino acids involved in the interaction with the RNA genome during encapsidation. We will determine if specific regions of the RNA genome are important for encapsidation. The long term goal of these studies will be to delineate the encapsidation signal for poliovirus, and elucidate the protein-protein and protein-nucleic acid interactions required for encapsidation. The results of these studies will provide insights into the encapsidation process which will lead to a more effective control of diseases associated with picornaviruses (and related RNA viruses), as well as information critical for the continued development and use of poliovirus as a vaccine vector.